The present invention relates to equipment and methods used in complex protein mixture analysis by 2-dimensional electrophoresis.
For any type of detailed substance analysis, a homogeneous sample of the substance of interest is required. For this reason, isolating a substance of interest from a mixture of substances is often necessary in many biochemical laboratories. There are many ways to separate substances: on the basis of size by molecular sieve chromatography or SDS-PAGE, on the basis of binding properties by affinity chromatography, or by isoelectric points (the pH at which the substance has no net charge) by isoelectric focusing. Isoelectric focusing is particularly effective for analyzing microheterogeneous protein species or other species which differ slightly in their chemical. content.
Isoelectric focusing with an immobilized pH gradient (IPG), makes true isoelectric focusing possible and significantly improves the reproducibility of the spot distribution along the pH gradient axis of 2-D maps. IPG also makes it possible to focus basic proteins in the gel and to obtain
Electrophoresis devices are well known in the art. However, attempts to construct an apparatus which successfully analyzes basic proteins (for example those with pH between 8-12) in a simple, user-friendly manner have previously been unsuccessful. Previous xe2x80x9cface upxe2x80x9d (gel side up) systems required messy preparation and critical setup to effectively load the sample on the gel. Sample cups had to be placed perfectly perpendicular to the gel (despite rotational freedom) and at the perfect height (despite placement flexibility on the vertical axis) using click stops to provide sample contact with the gel yet avoid crushing it. Newer xe2x80x9cface downxe2x80x9d (gel side down) systems which are easier to load and run, such as that described in co-assigned and application Ser. No. 09/095,002, now issued as U.S. Pat. No. 6,113,736, the contents of which are hereby incorporated by reference as if recited in full herein, cannot successfully separate basic proteins. Therefore, the present invention provides an apparatus which allows for effective analysis of basic proteins in a compact, simple way.
In view of the foregoing, it is an object of the present invention to provide a sample loading system that is capable of separating basic proteins by isoelectric focusing on an immobilized pH gradient (IPG) in a xe2x80x9cface upxe2x80x9d system.
It is also an object of the present invention to provide a sample loading means that is user-friendly and relatively clean.
It is another object of the present invention to simplify sample loading on xe2x80x9cface upxe2x80x9d gels.
It is a further object of the present invention to provide a means for accurate and uncomplicated sample positioning on the vertical axis, thereby providing adequate but not excessive contact of the sample and sample cup with the gel.
It is additionally an object of the present invention to create a gel loading system which is adjustable to different length gels and which allows flexibility of sample cup placement.
It is another object of the present invention to reduce the volume of mineral oil required to perform isoelectric focusing on a single gel.
These and other objects are satisfied by the present invention which is directed to gel loading systems, methods, and associated containers which are configured to successfully load electrophoresis gels with samples of any pH. In particular, a first aspect of the present invention is directed toward a sample loading assembly for electrophoresis gels comprising a gel holder adapted to hold a gel, two electrode carriers and associated electrodes, a sample loading cup adapted to load the sample onto the upper surface of the gel, and a cover, wherein said electrode carriers are configured such that, in ordinary use, the electrodes will be in electrical connection with the top surface of the gel. Specific embodiments include variations on the electrode placement along the gel surface and methods of electrode connection with the electrophoresis apparatus.